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Vasohibin-2 (VASH2) is a homolog of vasohibin-1 and exhibits pro-angiogenic activity. We recently reported that VASH2 is expressed in certain ovarian cancers and promotes tumor growth through angiogenesis. To further demonstrate the effectiveness of molecular targeting of VASH2 for anticancer treatment, we applied in vivo delivery of siRNA targeting VASH2 (siVASH2) using atelocollagen to a xenograft model of ovarian cancer. We inoculated mice s.c. with DISS and SKOV-3, two representative human ovarian serous adenocarcinoma cell lines. When tumors were measurable, we initiated treatment with control or siVASH2 mixed with atelocollagen, which enveloped the whole tumor. Treatment with siVASH2 significantly inhibited s.c. tumor growth by abrogating tumor angiogenesis. We confirmed that expression of VASH2 mRNA in the tumor was downregulated by siVASH2 treatment. In addition, the siVASH2-treated tumor contained more blood vessels covered with pericytes, indicating that knockdown of VASH2 contributes to the normalization of tumor blood vessels. Based on these results, VASH2 may be a promising molecular target for ovarian cancer treatment.
Ovarian cancer is the second most common gynecological malignancy and is the prime cause of death in industrialized countries. Debulking surgery and adjuvant combination chemotherapy with platinum and taxane are fundamental treatments, showing initial complete responses in 80% of patients.[2, 3] However, many patients experience abdominal recurrence; numerous novel clinical strategies are being evaluated for treating ovarian cancers to improve outcomes compared to outcomes of standard chemotherapy. Angiogenesis is recognized as a principal hallmark of various cancers and is also thought to be a key process for ovarian cancer growth. A promising option for treating ovarian cancer may be anti-angiogenic therapy. Indeed, a mAb against vascular endothelial growth factor (VEGF), bevacizumab, showed clinical activity against ovarian cancer in a phase 3 clinical trial. Although the effectiveness of anti-angiogenic drugs is encouraging, resistance to anti-angiogenic therapy has been reported in several review articles.[8-10] To overcome these problems, novel strategies for anti-angiogenic therapy must be developed.
The vasohibin family includes vasohibin-1 (VASH1) and vasohibin-2 (VASH2). Vasohibin-1 is an intrinsic negative feedback regulator of angiogenesis, which is selectively induced in endothelial cells (ECs) by an angiogenesis stimulator, such as VEGF or basic fibroblast growth factor. Vasohibin-2 is a homolog of VASH1 and exhibits pro-angiogenic activity at the sprouting front of angiogenesis. To date, very few reports regarding the relationship between VASH2 and tumor angiogenesis are available in published work.[13-15] In our previous study, the knockdown of VASH2 by the stable transfection of a shRNA vector showed notable inhibition of tumor growth, peritoneal dissemination, and tumor angiogenesis in a murine xenograft model of ovarian cancer. Therefore, we hypothesized that gene silencing by exogenous administration of siRNA should be effective in a therapeutic model of ovarian cancer. However, the rapid degradation of siRNA and short duration of its biological action in vivo require efficient delivery technology. To overcome this limited stability, several useful delivery systems, such as nanoparticles, liposomes, and adeno-associated viral vectors, have been developed. Atelocollagen, a highly purified pepsin-treated type I collagen of calf dermis, is a system for delivering siRNA. Collagen is a fibrous protein in the connective tissue, and plays an important role in maintaining tissue and organ morphology. A collagen molecule has an amino acid sequence known as the telopeptide at both the N- and C-termini, which confers most of its antigenicity. When the telopeptide is removed by treatment with pepsin, atelocollagen with low antigenicity is obtained. Atelocollagen is already in clinical use for wound healing and vessel prosthesis, and as a hemostatic agent. It has been reported that atelocollagen increases cellular uptake and nuclease resistance, prolongs release of the siRNA in vivo, and displays low toxicity.
In this study, we applied in vivo delivery of siRNA targeting VASH2 (siVASH2) by atelocollagen in an s.c. xenograft model of ovarian cancer cell lines to evaluate its therapeutic significance in terms of tumor growth, tumor angiogenesis, and tumor vessel maturation.
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In this study, we showed that exogenous treatment with siVASH2 and atelocollagen biomaterial has a significant knockdown effect on the VASH2 gene and an antitumor effect by decreasing tumor angiogenesis in vivo. In addition, siVASH2-treated tumors contained a larger number of mature blood vessels. These results suggest that VASH2 can be used as a novel molecular target for anti-angiogenic cancer therapy in ovarian cancer.
RNA interference technology using siRNAs has been experimentally introduced worldwide as a cancer therapy and is expected to be developed as a nucleic acid-based medicine. To obtain a sufficient anticancer effect, it is important not only to select appropriate gene targets, but also to develop suitable drug delivery systems (DDS). Atelocollagen is one such DDS of siRNA, and its usefulness in in vivo cancer models has been reported previously.[23-25] Several clinical trials for cancer therapy using RNAi techniques are also ongoing. However, to date, there has been no report on the use of DDS of siRNAs in gynecological malignancies, including ovarian cancer. Here, we showed the in vivo antitumor effect of RNAi technology targeting VASH2 with atelocollagen biomaterial in ovarian cancer for the first time.
Vasohibin-2 has been reported to be expressed in several types of cancers and acts on neighboring ECs as an angiogenesis stimulator in a paracrine manner. Specific knockdown of VASH2 from cancer cells retarded tumor growth by decreasing tumor angiogenesis, the mechanism of which is to inhibit proliferation and migration of ECs.[13-15] In this study, knockdown of endogenous VASH2 by treatment with siVASH2 and atelocollagen biomaterial retarded s.c. tumor growth by decreasing angiogenesis; a previous study used stable VASH2 knockdown clones to achieve this effect.[13-15] Treatment with siVASH2 alone did not significantly alter the in vitro proliferation of cancer cells. Expression of angiogenesis-related factors, including VEGF, TGFβ-1, and PDGF-BB, did not change following siVASH2 treatment. Therefore, knockdown of VASH2 may have indirectly affected tumor cells by inhibiting EC function. Our results showed that the knockdown efficacy of VASH2 considerably differed between in vitro and in vivo treatments. The in vivo knockdown effect may vary among cancer cells because in vivo tumors are heterogeneous; additionally, contamination with cells other than the cancer cells present in the tumor microenvironment may have occurred. Therefore, the discrepancy between the knockdown efficacy of VASH2 and angiogenesis inhibition may have been observed in in vivo experimental models, as shown in Figures 3-5.
vNewly formed tumor blood vessels are immature and show high vascular permeability, high interstitial pressure, and impaired blood flow. These characteristics cause resistance to conventional chemotherapy. Vascular normalization by pericyte coverage improves the tumor microenvironment, including blood flow, and facilitates chemotherapeutic drug delivery to the tumor tissue. Although anti-VEGF therapy such as bevacizumab normalizes tumor vessels, this effect is transient and tumor vessels eventually regress. Tumor vascular regression causes hypoxia within the tumor, followed by recurrence of tumor angiogenesis due to the development of compensatory mechanisms for producing angiogenic factors other than VEGF or the recruitment of bone marrow-derived angiogenic cells. Hypoxia due to the regression of tumor vessels could also make cancer cells more invasive and metastatic by inducing hypoxia-inducible factor-1. Therefore, persistent normalization of tumor vessels is required for improving the tumor microenvironment and inhibiting metastasis. Our results indicate that siVASH2-treated tumors contained more blood vessels covered with pericytes, suggesting that inhibition of VASH2 contributes to tumor vessel normalization. Although angiopoietins are strongly related to pericyte coverage, their primary expression level was low and siVASH2 treatment did not affect their expression level in cultured ovarian cancer cells. Thus, VASH2 might contribute to the destabilization of tumor blood vessels, which requires further investigation.
Vasohibin-2 is a promising molecular target for anti-angiogenic therapy that can be used to avoid the toxic side-effects of anti-VEGF therapy, such as hypertension and proteinuria, as it is a VEGF-independent and EC-extrinsic angiogenesis regulator and is expressed at low levels in normal adult tissues, except the brain and genital organs. Moreover, atelocollagen itself shows neither toxicity nor antigenicity in animals because antigenic telopeptides are removed through pepsin digestion. Collectively, RNAi therapy targeting VASH2 with atelocollagen biomaterial may be a powerful strategy against ovarian cancer and may offer safer and more favorable therapeutic outcomes. In addition, VASH1 inhibits tumor angiogenesis and makes tumor blood vessels mature. Moreover, VASH1 maintains the tumor vessels and vascular regression does not occur.[31, 32] Thus, a combination of VASH2 inhibition and VASH1 upregulation would be an attractive therapeutic strategy for cancer treatment.
To date, it has been shown that VASH1 inhibits tumor angiogenesis,[32-36] whereas VASH2 stimulates this process.[13-15] However, the putative vasohibin receptor and its downstream signaling have not been identified. It has been hypothesized that these two factors share a vasohibin receptor: one acts agonistically, and the other acts antagonistically. Studies are currently underway to resolve this question. Additionally, we are now searching for the bioactive center of VASH2 and attempting to develop a novel targeted molecular therapy such as a neutralizing mAb therapy. The significance and functional differences among splicing variants of VASH2 are also under investigation.
In conclusion, exogenous treatment using siVASH2 with atelocollagen biomaterial exerted a significant antitumor effect on ovarian cancer in vivo against tumor growth and tumor angiogenesis. Knockdown of VASH2 may also help tumor vascular normalization. Targeting VASH2 can be applied as a novel molecular target therapy for ovarian cancer.